Of great importance to man is the control of fungi which can cause human, animal and plant diseases as well as food spoilage. Considerable research and resources have been devoted to identifying antifungal agents. While certain methods and chemical compositions have been developed which aid in inhibiting or controlling the growth of fungi, new methods and antifungal compositions are needed.
Only about 100 of the thousands of known species of yeasts and molds cause disease in humans or animals. Only the dermatophytes and Candida are commonly transmitted from one human to another.
Human mycotic infections may be grouped into superficial, subcutaneous, and deep (or systemic) mycoses. Superficial fungal infections of skin, hair, and nails may be chronic and resistant to treatment but rarely affect the general health of the patient. Deep mycoses, on the other hand, may produce systemic involvement and are sometimes fatal.
The deep mycoses are caused by organisms that live free in nature in soil or on decaying organic material and are frequently limited to certain geographic areas. In such areas, many people acquire the fungal infection. A majority develop only minor symptoms or none at all, and only a small minority of infections progress to full-blown serious or fatal disease. The host""s cell-mediated immune reactions are of paramount importance in determining the outcome of such infections.
Pathogenic fungi generally produce no toxins. In the host, they regularly induce hypersensitivity to their chemical constituents. In systemic mycoses, the typical tissue reaction is a chronic granuloma with varying degrees of necrosis and abscess formation.
Fungal infections are common to a large number of animal species. Common agents of fungal infections include various species of the genii Candida and Aspergillus, and types thereof, as well as others. While external fungus infections can be relatively minor, systemic fungal infections can give rise to serious medical consequences. The incidence of fungal infections has undergone a significant increase, particularly in humans. This increase is, at least in part, attributable to an ever increasing number of patients having impaired immune systems, both as a result of medical therapy for other conditions, and as a result of diseases such as AIDS which compromise the immune system. Fungal disease, particularly when systemic, can be life threatening to patients having an impaired immune system. See, for example, U.S. Pat. No. 5,891,861.
A number of prior art pharmaceutical agents have been developed for the treatment of fungal diseases. These materials include compounds such as amphotericin B (AMB), triazoles and flucytosin. AMB is the drug of choice for many systemic fungal infections due to its broad range of activity; however, it is harmful to the kidneys and must be administered intravenously. Many of the triazoles exhibit broad ranging activity and can be administered orally; however, many strains of fungi have become resistant to these materials. Consequently, there is a need for a new group of agents which are effective in elminating fungus disease.
Food spoilage is typically caused by bacteria and fungi. Foods such as low-fat spreads, cheese, tea-based beverages, fruit- and tomato-based products are among the vulnerable food products. See, for example, U.S. Pat. No. 5,888,504. Although fungi can sometimes be controlled through heat treatment, an inactivating heat treatment is not always desirable or possible. Furthermore fungal spores present in factories can cause problems at the packing stage. Combating bacteria is relatively easy; fungi, however, can survive under very adverse conditions. Therefore, new compounds for preserving and protecting food are needed.
Post-harvest losses during storage of plant produce are caused, inter alia, by fungal and bacterial pathogens. Fungicidal compounds have long been used to increase yields and extend agricultural production capabilities into new areas. They have also been extremely important tools for ameliorating season-to-season differences in yield and quality caused by weather-driven variations in disease pressure.
Chemical fungicides have provided an effective method of control; however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water and the environment. Stringent new restrictions on the use of chemicals and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling fungi.
One example of the need to control post-harvest spoilage of agriculture products pertains to green and blue molds of citrus fruits caused by Penicillium digitatum and P. italicum. These molds cause severe damage during storage and shipping. The existing fresh-market industry relies completely on a combination of several chemical treatments to deliver sound fruit to distant markets over substantial periods of time without excessive damage caused by these molds. Unfortunately, there are increasing concerns about the safety of the chemicals currently used to control these fungal pathogens. Also, there are increasing problems with fungal strains with resistance to the most effective compounds.
In another example, powdery mildew of grapes caused by Uncinula necator can cause severe damage even in dry areas such as California. Traditionally this disease was controlled with applications of elemental sulfur, but this necessitates frequent, high volume applications of an irritating material. The introduction of egosterol biosynthesis inhibiting fungicides (primarily triazoles) greatly simplifies control, but also selects for tolerant strains. Some of these compounds are also known to have potential teratogenic effects and very long soil residuals. In these and other examples, alternative control methods are in great demandxe2x80x94particularly methods which are safer or more environmentally benign.
To prevent fungal spoilage it is common practice in many countries to spray produce with systemic fungicides in the field and to dip harvested produce in fungicide solutions prior to storage. Since the oncogenic nature of many of the most commonly used fungicides is increasingly recognized and because the persistence of most fungicides is increased by the low storage temperatures the postharvest use of fungicides is of growing concern.
Additionally, resistance to the fungicides, used has been reported and suppression of the main spoilage organism B. cinera by fungicides such as BENOMYL fungicide has been shown to result in increased population of A. brassicicola which causes a more penetrating rot of produce than B. cinera. See, for example, U.S. Pat. No. 5,869,038.
The future role of fungicides in agriculture is increasingly threatened by several factors including; the development of pest resistance, increasing concerns about food safety, and environmental accumulation of toxic compounds. As older fungicides are removed from the market due to regulatory changes there is an increasing need to find new effective fungicidal compounds.
In searching for new biologically active compounds, it has been found that some natural products and organisms are potential sources for chemical molecules having useful biological activity of great diversity. For example, the diterpene commonly known as taxol, isolated from several species of yew trees, is a mitotic spindle poison that stabilizes microtubules and inhibits their depolymerization to free tubulin (Fuchs, D. A., R. K. Johnson [1978] Cancer Treat. Rep. 62:1219-1222; Schiff, P. B., J. Fant, S. B. Horwitz [1979] Nature (London) 22:665-667). Taxol is also known to have antitumor activity and has undergone a number of clinical trials which have shown it to be effective in the treatment of a wide range of cancers (Rowinski, E. K. R. C. Donehower [1995] N. Engl. J. Med. 332:1004-1014). See also, e.g., U.S. Pat. Nos. 5,157,049; 4,960,790; and 4,206,221.
Marine sponges have also proven to be a source of biologically active chemical molecules. A number of publications disclose organic compounds derived from marine sponges including Scheuer, P. J. (ed.) Marine Natural Products, Chemical and Biological Perspectives, Academic Press, New York, 1978-1983, Vol. I-V; Uemura, D., K. Takahashi, T. Yamamoto, C. Katayama, J. Tanaka, Y. Okumura, Y. Hirata (1985) J. Am. Chem. Soc. 107:4796-4798; Minale, L. et al. (1976) Fortschr. Chem. org. Naturst. 33:1-72; Faulkner, D. J. (1998) Natural Products Reports 15:113-158; Gunasekera, S. P., M. Gunasekera, R. E. Longley and G. K. Schulte (1990) xe2x80x9cDiscodermolide: A new bioactive polyhydroxy lactone from the marine sponge Discodermia dissolutaxe2x80x9d J. Org. Chem., 55:4912-4915; (1991) J. Org. Chem. 56:1346; U.S. Pat. No. 4,808,590 discloses compounds, having antiviral, antitumor, and antifungal properties, isolated from the marine sponge Theonella sp. New antiviral, antitumor and antifungal compositions and their methods of use are described in U.S. Pat. Nos. 4,801,606; 4,808,590, T. Higa, S. Sakemi and S. Cross. Also, the isolation and elucidation of spongistatins 1-7 are described in U.S. Pat. Nos 5,328,929; 5,393,897; and 5,436,400. These compounds have been found to have antitumor and antifungal properties. See, U.S. Pat. No. 5,883,120.
Other natural products of marine origin include the following:
1. Plakinic acid A 
Source: unnamed sponge of the family Plakinidae from Caribbean
Reference: Phillipson, D. W.; Rinehart, K. L. Jr. J. Am. Chem. Soc. 1983, 105, 7735-7736.
Antifungal activity: 24 mm vs. S. cerevisiae and 25 mm vs. P. atrovenetum at 100 mg/disk
2. Plakinic acids C and D and epi-plakinic acid C and D 
Source: Plakortis sp. collected in the Fiji Islands
Reference: Davidson, B. S. J. Org. Chem. 1991, 56, 6722-6724.
Cytotoxicity: against human epidermoid carcinoma (KB) cells, human colorectal adenocarcinoma (LoVo) cells, and L1210 murine leukemia cells.
3. Epiplakinic acid E methyl ester 
Source: Plakinastrella onkodes collected in the Gulf of Mexico
Reference: Horton, P. A.; Longley, R. E.; Kelly-Borges, M.; McConnell, O. J.; Ballas, L. M. J. Nat. Prod. 1994, 57, 1374-1381.
Cytotoxicity: against human lung carcinoma (A549) and murine leukemia (P388) cells.
4. (3R,5S,12E,14E,17Z)-3,5-dimethyl-3,5-peroxydodeca-12,14,17-trienoate and methyl ester 
Source: Plakinastrella sp. collected at Hagakhak Island, Philippines
Reference: Qureshi, A.; Salva, J. Harper, M. K.; Faulkner, D. J. J. Nat. Prod. 1998, 61, 1539-1542.
Essentially inactive against Candida albicans 
5. Natural and unnatural 1,2-dioxolane carboxylate analogues 
Rxe2x95x90C13H27 
Rxe2x95x90C14H29 
Rxe2x95x90C15H31 
Rxe2x95x90C16H33 
Rxe2x95x90C17H35 
Source: isolated from Halichondriidae sponges or prepared by synthesis
Reference: Patil, A. D. U.S. Pat. No. 4,879,307 (1989); C. A., 1988, 109, 17027f.
Reference: Bloodworth, A. J.; Bothwell, B. D.; Collins, A. N.; Maidwell, N. L. Tetrahedron Let. 1996, 37, 1885-1888. (Synthesis)
Activity: Active against tumor cell lines
6. Plakortin and its free acid; 3-Epiplakortin; 9,10-dihydro-3-epiplakortin; and other related skeleton peroxides 
Source: Caribbean sponge Plakortis halichondrioides and/or Plakortis zyggompha 
Reference: Higgs, M. D.; Faulkner, D. J. J. Org. Chem. 1978, 43, 3454-3457.
Reference: Stierle, D. B.; Faulkner, D. J. J. Org. Chem. 1979, 44, 964-968.
Reference: Stierle, D. B.; Faulkner, D. J. J. Org. Chem. 1980, 45, 3396-4301.
Reference: Phillipson, D. W.; Rinehart, K. L. Jr. J. Am. Chem. Soc. 1983, 105, 7735-7736.
Activity: plakortin was essentially bioinactive, the free acid was shown to inhibit S. cerevisiae, P. atrovenetum and B. subtilis. The activity of the other compounds was not reported.
7. Two epimeric peroxy acids and their esters 
Source: Carribbean sponge Chondrosia collectrix 
Reference: Stierle, D. B.; Faulkner, D. J. J. Org. Chem. 1979, 44, 964-968.
Activity: Four compounds were reported as having mild antibacterial activity
8. Plakinic acid B 
Source: unnamed sponge of the family Plakinidae from the Caribbean
Reference: Phillipson, D. W.; Rinehart, K. L. Jr. J. Am. Chem. Soc. 1983, 105, 7735-7736.
Activity: 20 mm vs. S. cerevisine and 18 mm vs. P. atrovenetum at 100 mg/disk
9. Unsaturated cyclic-peroxide-containing acids 
Source: Plakortis angulospiculatus collected off the coast of Venezuela
Reference: Gunasekera, S. P.; Gunasekera, M.; Gunawardana, G. P.; McCarthy, P.; Burres, N. J. Nat. Prod. 1990, 53, 669-674.
Activity: 1.6 mg/ml against Candida albicans, and were also active against Aspergillus nidulans and Bacillus subtilis. Cytotoxicity gave IC50""s of 0.2-0.9 mg/ml against P388 cells.
10. Cyclic-peroxide-containing acid 
Source: Plakortis halichondrioides collected off the coast of Jamaica
Reference: Rudi, A.; Kashman, Y. J. Nat. Prod. 1993, 56, 1827-1830.
Activity: displayed an IC50 value against P388 murine leukemia of 0.5 mg/ml.
11. Plakortide F, G, H 
Source: Plakortis halichondrioides collector in Jamaica
Reference: Patil, A. D.; Freyer, A. J.; Carte, B. Johson, R. K.; Lahouratate, P. J. Nat. Prod. 1996, 59, 219-223.
Activity: significantly enhanced Ca2+ uptake by the cardiac sarcoplasmic reticulum.
12. Cyclic polyketide peroxides 
Source: Okinawan Plakortis sp.
Reference: Fontana, A.; Ishibashi, M.; Kobayashi, J. Tetrahedron. 1998, 54, 2041-2048.
Reference: Fontana, A.; Ishibashi, M.; Shigemori, H.; Kobayashi, J. J. Nat. Prod. 1998, 61, 1427-1429.
Activity: Cytotoxicity against human epidermoid carcinoma KB and murine lymphoma L1210 cells was reported.
13. Cyclic peroxides 
Source: Palauan sponge of Plakortis aff. angulospiculatus
Reference: Compagnone, R. S.; Pina, I. C.; Rangel, H. R.; Dagger, F.; Suarez, A. I.; Reddy, M. V. R.; Faulkner, D. J. Tetrahedron, 1998, 54, 3057-3068.
Activity: showed in vitro antiproliferative effects on promastigotes of Leishmania mexicana, a flagellate protozoan that causes leishmaniasis.
14. Polyketide peroxide 
Source: Plakortis sp. collected on the west rim of St. Francis Atoll, Amirante Islands
Reference: Braekman, J. C.; Daloze, D.; Groote, S. D.; Fernandes, J. B.; Van Soest, R. W. M. J. Nat. Prod. 1998, 61, 1038-1042.
Activity: exhibited toxicity toward Artemia larvae (LD50 15 mg/L).
15. Cyclic polyketide peroxides 
Source: Plakortis lita from Papua New Guinea
Ref: Harrison, B.; Crews, P. J. Nat. Prod. 1998, 61, 1033-1037.
Activity: activity in vitro against solid tumor and L1210 leukemia cell lines was reported.
Also, U.S. Pat. No. 4,731,377 describes cyclic peroxides having antitumor properties.
A principal object of the subject invention is the provision of novel compositions of biologically active compounds. These compounds have been found to have antifungal activity. Because of this antifungal activity, the compounds of the subject invention can advantageously be used for control of unwanted fungi such as those which cause human, animal and plant disease, and those that cause spoilage of food, cosmetics, and other consumer items.
Specifically exemplified herein are six compounds (Compounds 1-6), shown below, which have been found to possess antifungal activity. The compounds of the subject invention have a cyclic peroxide functionality forming either a six or a five membered ring. The compounds also have a free carboxylic acid moiety adjacent to the peroxide-containing ring.
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
The structures of 1-4 and 6 have not been previously disclosed, while the structure of 5 has been disclosed (Longley, NMHCC Conference on Natural Products Drug Discovery, Nov. 12-14, 1997, Baltimore, Md.), but not as an antifungal agent.
Compounds 1-4 are significantly different from previously reported compounds. Compound 5 has been reported previously as an inhibitor of the enzyme cdc25a, but has not been reported to have antifungal activity.
All of the compounds have utility as antifungal agents. As such the compounds of the subject invention can be used as pharmaceutical agents to treat fungal infections in humans or animals. The compounds can also be used as disinfectants, food preservatives, and to treat plant diseases caused by fungi.
In accordance with the subject invention, methods for inhibiting fungi in a host include contacting the fungi with an effective amount of the compositions of the subject invention.
Additional aspects of the invention include the provision of methods for producing the new compounds and compositions.
Other objects and further scope of applicability of the present invention will become apparent from the detailed descriptions given herein; it should be understood, however, that the detailed descriptions, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent from such descriptions.